1. Field of the Invention
The present invention concerns the utilization of addition polymerization, e.g., vinyl polymerization, in diagnostic test compositions for detecting and measuring an analyte possessing biologic activity and in methods for detecting and measuring an analyte possessing biologic activity.
2. Background Information
Modern diagnostic tests for trace amount of analyte possessing biologic activity, such as those encountered in clinical laboratory medicine often employ immunologic (i.e., antigen-antibody) reactions.
Comparable techniques employing nucleic acid hybridization probes for the analysis of nuclei acids have entered the realm of clinic and research laboratories.
These highly specific reactions are detected by means of a label or tag, attached to one of the components, that is either chromophoric, fluorescent, radioactive, chemiluminescent or an enzyme that can generate such signals. Chromophore labels for detection of trace analytes are of historic and academic interest only because of their low sensitivity. Color detection methods are capable of detecting no fewer than 10.sup.18 to 10.sup.15 analyte molecules per milliliter, while radioisotope detection methods can approach a detection sensitivity in the range of 10.sup.8 to 10.sup.7 analyte molecules per milliliter.
Fluorescent chemiluminescent and enzymatic methods as characterized by relative-specific-activities can be more sensitive than radiosiotope methods by at least two or more magnitudes. These are goals, however, and not achievements. Continued increases in sensitivity are reported as these methods are refined, but no magnitude jumps are anticipated.
Prior to the present invention, no one proposed the coupling of an extraneous non-catalytic substance with a catalyst component of vinyl polymerization. If the extraneous substance is a highly specific ligand for a target analyte of interest, then the production of detectable polymer can indicate the presence and site of the analyte. This linkage provides the basis of a unique detection system. The present invention when applied to analytes of biologic interest provides a diagnostic technique useful in the fields of medicine and agriculture.
The present invention is designed to detect and measure the following types of analytes:
1. An analyte that possesses photocatalyst properties for the production of addition, e.g., vinyl, polymers; PA1 2. an analyte that does not possess such photocatalyst properties, but forms a stable complex with a specific ligand, the latter capable of being linked to one or more moieties of such a catalyst system. Such analytes include, among others, immunoreactive substances and nucleic acids. PA1 (a) a photocatalyst system capable of converting a monomer to a polymer on exposure to light, said monomer capable of undergoing addition polymerization, the photocatalyst system comprising one or more chemical moieties with PA1 (b) at least one monomer capable of undergoing addition polymerization. PA1 (a) combining 1) the photosensitizer analyte or 2) the enzyme analyte that generates a photosensitizer from its precursor, the photosensitizer in the presence of an electron donor capable on exposure to light of converting a monomer to a polymer through addition polymerization, said monomer capable of undergoing addition polymerization, 3) an electron donor and 4) in the case of the enzyme a precursor of the photosensitizer together with 5) at least one monomer capable of undergoing addition polymerization, PA1 (b) exposing the resultant combination of (a) to light and PA1 (c) determining the extent of addition polymerization as an indicator of the presence and/or quantity of analyte. PA1 (a) combining 1) the electron donor analyte or 2) the enzyme analyte that generates an electron donor from its precursor, the electron donor capable of reducing a light excited photosensitizer, the photosensitizer capable of converting a monomer to a polymer through addition polymerization, said monomer capable of undergoing addition polymerization, 3) a photosensitizer and, 4) in the case of the enzyme, a precursor of the electron donor, together with 5) at least one monomer capable of undergoing addition polymerization, PA1 (b) exposing the resultant combination of (a) to light and PA1 (c) determining the extent of addition polymerization as an indicator of the presence and/or the quantity of analyte. PA1 (a) linking an antigen or a hapten to 1) a photosensitizer or 2) an enzyme that generates a photosensitizer from its precursor, the photosensitizer capable of converting a monomer capable of undergoing addition polymerization, e.g., a vinyl monomer or a vinylidine monomer, to a polymer, e.g., a vinyl polymer, on exposure to light, PA1 (b) contacting the linked antigen or hapten with a complementary antibody or an antigen binding segment of an antibody in the presence of an electron donor and in the case of the enzyme, a precursor of a photosensitizer, together with at least one monomer capable of undergoing addition polymerization, PA1 (c) exposing the resultant mass from (b) to light, and PA1 (d) determining the extent of addition polymerization as an indicator of the presence and/or the quantity of the antibody or antigen binding segment of an antibody. PA1 (a) linking an antibody or an antigen binding segment of an antibody to (1) a photosensitizer or (2) an enzyme that generates a photosensitizer from its precursor, the photosensitizer in the presence of an electron donor capable of converting a monomer capable of undergoing addition polymerization, e.g., a vinyl monomer or a vinylidine monomer to polymer e.g., a vinyl polymer, on exposure to light. PA1 (b) contacting the linked antibody or an antigen binding segment of an antibody with a complementary antigen or hapten in the presence of an electron donor and in the case of the enzyme, a precursor of a photosensitizer together with at least one monomer capable of undergoing addition polymerization, PA1 (c) exposing the resultant mass from (b) to light, and PA1 (d) determining the extent of addition polymerization as an indicator of the presence and/or the quantity of the antigen or hapten. PA1 (a) linking an antigen or a hapten to (1) an electron donor or (2) an enzyme that generates an electron donor from its precursor, the electron donor being capable of reducing a light excited photosensitizer, the photosensitizer capable of converting a monomer capable of undergoing addition polymerization, e.g., a vinyl monomer or a vinylidine monomer, to a polymer, e.g., a vinyl polymer, on exposure to light, PA1 (b) contacting the linked antigen or hapten with a complementary antibody or an antigen binding segment of an antibody in the presence of a photosensitizer and in the case of the enzyme, a precursor of an electron donor, together with at least one monomer capable of undergoing addition polymerization, PA1 (c) exposing the resultant mass from (b) to light, and PA1 (d) determining the extent of addition polymerization as an indicator of the presence and/or the quantity of the antibody or an antigen binding segment of an antibody. PA1 (a) linking an antibody or an antigen binding segment of an antibody to (1) an electron donor or (2) an enzyme that generates an electron donor from its precursor, the electron donor capable of reducing a light excited photosensitizer, the photosensitizer capable of converting a monomer capable of undergoing addition polymerization, e.g., a vinyl monomer or a vinylidine monomer to a polymer, e.g., a vinyl polymer, on exposure to light, PA1 (b) contacting the linked antibody or an antigen binding segment of an antibody with a complementary antigen or hapten in the presence of a photosensitizer and in the case of the enzyme, a precursor of an electron donor together with at least one monomer capable of undergoing addition polymerization, PA1 (c) exposing the resultant mass from (b) to light, and PA1 (d) determining the extent of addition polymerization as an indicator of the presence and/or the quantity of the antigen or hapten. PA1 (a) linking a hybridizable nucleic acid probe containing a known sequence, said sequence being single stranded and complementary to a single stranded segment of an analyte nucleic acid to 1) a photosensitizer or 2) an enzyme that generates a photosensitizer from its precursor, the photosensitizer capable of converting a monomer capable of undergoing addition polymerization, e.g., a vinyl monomer or a vinylidine monomer to a polymer, e.g., a vinyl polymer, on exposure to light, PA1 (b) contacting the linked nucleic acid probe with the analyte nucleic acid in the presence of an electron donor and in the case of the enzyme, a precursor of a photosensitizer, together with at least one monomer capable of undergoing addition polymerization, PA1 (c) exposing the resultant mass from (b) to light and PA1 (d) determining the extent of addition polymerization as an indicator of the presence of and/or the quantity of nucleic acid analyte. PA1 (a) linking a hybridizable nucleic acid probe containing a known sequence, said sequence being single stranded and complementary to a single stranded segment of an analyte nucleic acid to 1) an electron donor or 2) an enzyme that generates an electron donor from its precursor, the electron donor being capable of reducing a light excited photosensitizer, the photosensitizer capable of converting a monomer capable of undergoing addition polymerization, e.g., a vinyl monomer or a vinylidene monomer, to a polymer, e.g., a vinyl polymer, on exposure to light, PA1 (b) contacting the linked nucleic acid probe with the analyte nucleic acid in the presence of a photosensitizer and in the case of an enzyme, a precursor of an electron donor, together with at least one monomer capable of undergoing addition polymerization, PA1 (c) exposing the resultant mass from (b) to light, and PA1 (d) determining the extent of addition polymerization as an indicator of the presence and/or the quantity of nucleic acid analyte. PA1 (a) contacting a hybridizable nucleic acid probe containing a known sequence, said sequence being single stranded and complementary to a single stranded segment of nucleic acid analyte with said analyte, PA1 (b) linking the probe to 1) a photosensitizer or 2) to an enzyme that generates a photosensitizer from its precursor, the photosensitizer capable of converting a monomer capable of undergoing addition polymerization, e.g., a vinyl monomer or a vinylidine monomer, to a polymer, e.g., a vinyl polymer, on exposure to light, in the presence of an electron donor and in the case of an enzyme, a precursor of a photosensitizer, together with at least one monomer capable of undergoing addition polymerization, PA1 (c) exposing the resultant mass from (b) to light and PA1 (d) determining the extent of addition polymerization as an indicator of the presence and/or quantity of nucleic acid analyte. PA1 (a) contacting a hybridizable nucleic acid probe containing a known sequence, said sequence being single stranded and complementary to a single stranded segment of a nucleic acid analyte, with said analyte, PA1 (b) linking the probe to 1) an electron donor or 2) to an enzyme that generates an electron donor from a precursor, the electron donor being capable of reducing a light excited photosensitizer, the photosensitizer capable of converting a monomer capable of undergoing addition polymerization, e.g., a vinyl monomer or a vinylidine monomer, to a polymer, e.g., a vinyl polymer, on exposure to light, in the presence of a photosensitizer and, in the case of an enzyme, a precursor of an electron donor, together with at least one monomer capable of undergoing addition polymerization, PA1 (c) exposing the resultant mass from (b) to light, and PA1 (d) determining the extent of addition polymerization as an indicator of the presence and/or quantity of nucleic acid analyte. PA1 (1) The photosensitizer of a photocatalyst system for vinyl polymerization can be recycled indefinitely in the presence of an electron donor source that protects the photosensitizer from undergoing irreversible photooxidation. Each cycle of photoreduction yields a free radical capable of initiating chain polymerization. PA1 (2) The photosensitizer and/or the electron donor can be generated by an enzyme-labeled analyte. Horseradish peroxidase is one such enzyme. PA1 (3) A single free radical molecule can initiate the formation of a polymer chain molecule composed of 10.sup.5 to 10.sup.6 monomer molecule units. A particle visible to the naked eye under light scattering conditions can be the product of as few as 2 to 4 free radical molecules.